A RIA for mouse placental lactogen-I (mPL-I) was developed using recombinant mPL-I as the standard, radioligand, and antigen for antiserum production. Displacement curves for dilutions of serum and placental extracts from pregnant mice were parallel to the recombinant mPL-I standard curve. Serum from male and nonpregnant female mice and high concentrations of mouse PRL, GH, PL-II, proliferin, and proliferin-related protein did not cross-react in the assay. mPL-I appeared in maternal serum on day 6 of pregnancy. Its concentration remained low until day 8 and them increased to a very large peak on days 9-11 (maximum concentration, approximately 8 micrograms/ml). The mPL-I concentration declined after day 11, but the hormone could be detected at low concentration in maternal serum until the end of pregnancy. On day 10 of pregnancy, the mPL-I concentration of maternal serum was correlated with litter size. Fractionation of serum from 10-day pregnant mice by size exclusion chromatography indicated the absence of high mol wt forms of mPL-I in the circulation.
Two 5'-untranslated regions (5'UTRs) with distinctly different sequences, designated 5'UTR L1 and 5'UTR L2, were obtained by amplification of complementary DNA from mouse liver and placenta with primers complementary to sequences from the hormone-binding domain common to GH receptor (GHR) and GH-binding protein (GHBP) messenger RNAs (mRNAs). The presence of an open reading frame in the 5'UTR L2 and the high GC content of this sequence suggest that mRNAs containing this 5'UTR may be translated with a lower efficiency than those containing 5'UTR L1. Expression studies showed that 5'UTR L1 and 5'UTR L2 are present in GHR and GHBP mRNAs in both tissues. However, the relative expression of the two 5'UTRs differs between liver and placenta and in liver from different physiological states. The different expression patterns of the L1 and L2 5'UTRs predict that the corresponding 5'-noncoding exons of the GHR/GHBP gene are associated with different regulatory elements. The expression patterns of the 5'UTRs also indicate that there is a linkage between the 5'UTR present in GHR/GHBP gene transcripts and the alternative splicing of these transcripts to yield either GHR or GHBP mRNAs. The 5'-noncoding exon used for transcription of the GHR/GHBP gene, therefore, may be involved in regulating both the ratio of GHR to GHBP transcripts and the efficiency of translation of these transcripts. Transcription from the different 5'-noncoding exons of the GHR/GHBP gene thus may be a critical element in the regulation of the expression of GHR and GHBP and thereby in the control of the responses of different tissues to GH.
The effects of secretagogue(s) from mouse decidual tissue on the release of mouse placental lactogen-II (mPL-II) were studied. Decidual tissue was obtained from 10- and 11-day-pregnant mice. The tissue was homogenized, extracted, and the tissue extract was made 50% saturated with ammonium sulfate. Both the precipitate and supernatant were tested for their ability to stimulate mPL-II release from cultured trophoblasts. The supernatant contained an activity to stimulate the release of mPL-II. This activity was further purified using column chromatography. The purification resulted in isolation of a protein with a mol wt of 20 K as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and 6 K under reducing conditions. Further characterization of this protein showed that it binds calcium and has an amino acid sequence that is highly homologous with calcyclin expressed in mouse embryonic fibroblast cells and with calcyclin from other species. This protein was designated mouse decidual calcyclin. Antiserum was raised against the purified decidual calcyclin for development of an RIA and for immunoblots. Western blots of various mouse tissue extracts and mouse serum from different physiological stages showed that the concentration of calcyclin was highest in decidual tissue. Detectable levels were found in extracts from trophoblast, lung, and stomach, but the concentrations in these tissues were about 100 times lower than in decidua. Decidual calcyclin was not detectable in mouse serum. Cultured decidual cells released calcyclin into the medium. On average, this release was about 7.8 ng/micrograms DNA.24 h. The rate of release did not change significantly during 4 days of culture. The ratio of calcyclin in cells per calcyclin released during 24 h averaged 2.3 and did not change significantly during the culture period. The purified decidual calcyclin stimulated the release of mPL-II from cultured trophoblasts in a dose-dependent manner at concentrations from 0.01 to 1 microgram/ml. The maximum stimulation averaged about 1.5 times above control. It is concluded that decidual calcyclin may be of physiological importance for the regulation of mPL-II secretion.
The mouse placenta produces several polypeptides belonging to the prolactin-growth hormone gene family, including mouse placental lactogen (mPL) I and mPL-II. The present study was undertaken to determine whether the secretion of mPL-I and mPL-H is regulated by interleukin 6 (IL-6), which is present in the placenta and has previously been reported to stimulate the secretion of pituitary members of this gene family. Effects of human and mouse IL-6 on mPL-I and mPL-II secretion were examined in primary cultures of placental cells from days 7, 9, and 12 of pregnancy. IL-6 caused a dose-dependent reduction in the mPL-HI concentration in the medium of cells from days 9 and 12 of pregnancy but did not affect the mPL-II concentration in the medium ofcells from day 7 of pregnancy or the mPL-I concentration in the medium of cells from days 7 or 9 of pregnancy. The lowest concentration of human IL-6 that significantly inhibited mPL-II secretion was 250 pM. The effect of IL-6 on the mPL-II concentration in the medium was due primarily to inhibition of mPL-II synthesis, which resulted at least partly from a decrease in the steady-state level of mPL-II mRNA. These data raise the possibility that IL-6 may regulate mPL-I production after midpregnancy in vivo.
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